The percentage of CD39+ monocytes is higher in pregnant COVID-19+ patients than in nonpregnant COVID-19+ patients

Current medical guidelines consider pregnant women with COVID-19 to be a high-risk group. Since physiological gestation downregulates the immunological response to maintain “maternal-fetal tolerance”, SARS-CoV-2 infection may constitute a potentially threatening condition to both the mother and the fetus. To establish the immune profile in pregnant COVID-19+ patients, a cross-sectional study was conducted. Pregnant women with COVID-19 (P-COVID-19+; n = 15) were analyzed and compared with nonpregnant women with COVID-19 (NP-COVID-19+; n = 15) or those with physiological pregnancy (P-COVID-19-; n = 13). Serological cytokine and chemokine concentrations, leucocyte immunophenotypes, and mononuclear leucocyte responses to polyclonal stimuli were analyzed in all groups. Higher concentrations of serological TNF-α, IL-6, MIP1b and IL-4 were observed within the P-COVID-19+ group, while cytokines and chemokines secreted by peripheral leucocytes in response to LPS, IL-6 or PMA-ionomicin were similar among the groups. Immunophenotype analysis showed a lower percentage of HLA-DR+ monocytes in P-COVID-19+ than in P-COVID-19- and a higher percentage of CD39+ monocytes in P-COVID-19+ than in NP-COVID-19+. After whole blood polyclonal stimulation, similar percentages of T cells and TNF+ monocytes between groups were observed. Our results suggest that P-COVID-19+ elicits a strong inflammatory response similar to NP-COVID19+ but also displays an anti-inflammatory response that controls the ATP/adenosine balance and prevents hyperinflammatory damage in COVID-19.


Introduction
Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus infections result in high mortality among pregnant women (25% and 27%, respectively) [1]. In 2019, a new coronavirus called SARS-CoV-2 appeared and became a new high-risk virus because SARS-CoV-2 viral infections lead to a powerful cell and humoral immune response in pregnant women and increase fetal morbidity and mortality [2][3][4]. Additionally, pregnant women exhibit higher mortality rates associated with viral infections than the general population [5,6]. The immune response in pregnant women is mediated by many cellular and humoral mechanisms [7,8], resulting in a unique biological scenario to face a new virus, as in COVID- 19. Comorbidities associated with critical COVID-19 are highly prevalent in Mexico in both the general population and pregnant women [3,4,9,10]; however, the clinical presentation of COVID-19 seems to be similar in both the general population and pregnant women [3,11]. Current data are not sufficient to determine whether vertical transmission is possible, how large the potential infection is by asymptomatic pregnant women to the general population, or whether COVID-19 leads to an increase in postpartum mortality. In addition, the epidemiological and clinical characteristics and immune profile of pregnant women with COVID-19 have been poorly explored. As it is in the general population, lymphopenia is also reported in pregnant women with COVID-19 [12]. Parameters such as neutrophil count are useful for COVID-19 diagnosis [13]. Furthermore, an increased neutrophil/lymphocyte ratio has been associated with fatal outcomes in COVID-19 patients [14][15][16][17]. Several studies indicate that leucocyte count is necessary for the initial evaluation of COVID-19 patients; however, a detailed phenotypic analysis of leucocytes could improve our knowledge of SARS-CoV-2 infection, especially in pregnant women.
The COVID-19-derived inflammatory response in pregnancy could disturb the delicate immune balance in cellular components. Some surface molecules on cells can modulate leucocyte activation and function and, in pregnancy, also trigger regulator systems that may prevent both fetal rejection and the inflammatory response. CD39 and CD73 are ectonucleotidases that metabolize ATP to adenosine, which drives immune profiles from proinflammatory to anti-inflammatory [27]. Additionally, CD39 downregulation is associated with poor pregnancy outcomes [28]. Analysis of CD39 and CD73 in COVID-19 patients showed a decrease in CD8+CD73+ T cells, and NKT cells correlated with ferritin levels and were potential useful prognostic markers in COVID-19 [29]. Additionally, lower plasma ATP and adenosine levels were identified in mild and severe COVID-19 patients, higher frequencies of CD4 +CD39+ cells in severe COVID-19 patients, and diminished frequencies of CD4+CD73+ and CD8+CD73+ cells in severe COVID-19 patients compared to mild COVID-19 patients and controls [30]. However, no evidence of the CD39/73 system has been reported in pregnant women with COVID-19.
To analyze the immune profile in P-COVID-19+, a cross-sectional study was conducted. Our analysis includes a) the immunophenotype of lymphocytes and monocytes expressing activation markers, b) serum cytokine/chemokine concentration, and c) the proportion of cytokine-producing leucocytes in response to polyclonal stimuli. Herein, we explored cellular and humoral characteristics that may improve the understanding of the immunopathophysiology of pregnant women with COVID-19.

Patients
This study was conducted by the "Servicio de Ginecología y Obstetricia" at the Hospital General de Mexico "Dr. Eduardo Liceaga" in conjunction with the Unidad de Investigación Médica en Inmunoquímica (UIMIQ) at the Hospital de Especialidades, Centro Médico Nacional Siglo XXI, and the Servicio de complicaciones de la segunda mitad del embarazo, División Obstetricia, UMAE Hospital de Gineco-Obstetricia No. 4 "Dr. Luis Castelazo Ayala". The study was evaluated by the National Committee for Scientific Research (CNIC) with the following approval numbers: Research project: DI / 20112/04/45, and R-2020-785-095. After obtaining a signed informed consent letter, forty-four women were enrolled and assigned to one of three groups: a) NP-COVID-19+, n = 15, b) P-COVID-19+, n = 15, and c) P-COVID-19-, n = 13. The COVID-19 diagnosis was based on clinical characteristics [31], and SARS--CoV-2 viral infection was confirmed by specific reverse transcription-polymerase chain reaction. Comorbidities and clinical features were recorded.

Blood sample collection
Our study is in accordance with the World Medical Association's Declaration of Helsinki. After a patient signed the informed consent letter, blood specimens were collected in siliconecoated tubes (EDTA or heparinized tubes, BD Vacutainer, N. J, USA). Samples were processed immediately after collection. FSC-H plot, and leucocytes were identified using an SSC vs. CD45 plot. Lymphocytes were gated as SSC low FSC low CD45 ++ CD14 -, monocytes as SSC mid FSC mid CD45 + CD14 + , and neutrophils as SSC mid FSC mid CD45 + CD16 + . Lymphocyte subtypes were identified according to CD19 + (B cells) and CD3+ (T cells), which could be CD4+ (Th) or CD8+ (Tc). The percentages and mean fluorescence intensities (MFIs) of HLA-DR, CD69, CD39, CD73, CD32 and CCR5 were calculated.

Intracellular cytokine detection
After cell culture, whole blood samples (10x10 5

Statistical analysis
Statistical analysis was performed using GraphPad Prism 1 version 7 software (GraphPad Software, San Diego, CA, USA). Nonparametric ANOVA (Kruskal-Wallis test) with Dunn's posttest was applied. Categorical variables were expressed as percentages (% and compared by Fisher's exact test. A p<0.05 was considered statistically significant.

Results
We compared P-COVID-19+, NP-COVID-19+ and clinically healthy pregnant women (P-COVID-19-) to assess the immune profile. Table 1 shows the demographic and clinical features. Pregnant women with or without COVID-19 had similar maternal and gestational ages. Additionally, the frequency of comorbidities (diabetes mellitus and systemic arterial hypertension) was similar among the groups. Some clinical characteristics, such as heart rate and serological lactate dehydrogenase (LDH) concentration, were higher in the P-COVID-19+ patients than in the P-COVID-19-patients (p = 0.048 and p = 0.005, respectively). No significant Table 1. Clinical and laboratory characteristics.

NP-COVID-19+ (n = 9-15)a P-COVID-19+ (n = 8-15)b P-COVID-19-(n = 13)c p
Age ( difference was observed between the P-COVID-19+ and P-COVID-19-patients for age, BMI, respiratory rate, body temperature, mean arterial pressure (MAP), hemoglobin, total leucocyte count, neutrophil/lymphocyte ratio (NLR), total platelet count, serum glucose, serum creatinine, partial thromboplastin time or fibrinogen. Diminished oxygen saturation levels were observed in the COVID-19+ pregnant and nonpregnant patients, but without significant differences among them. Regarding D-dimer concentrations, higher levels were observed in the P-COVID-19+ than in the NP-COVID-19+ patients; however, the highest D-dimer concentration was found in those patients with physiological pregnancy. We did not observe differences among groups in total leucocyte count or in subtypes of leucocytes identified by flow cytometry (S1 Table). However, the lowest percentage of lymphocytes was observed in the NP-CO-VID-19+ patients. Table 2 shows the frequency of symptoms in those patients with and without COVID-19. The pregnant women with COVID-19 had a similar frequency of symptoms such as cough, myalgia, arthralgia and dyspnea as the NP-COVID19+ group. The most frequent symptoms in the pregnant patients with COVID-19 were cough, myalgia, and arthralgia.

Discussion
During pregnancy, the immune system is highly regulated. Multiple mechanisms of immune tolerance develop during gestation and favor physiological progress in reproduction [8]. Any  [2]. However, we found no differences in clinical manifestations or laboratory values between P-COVID-19 + and P-COVID-19-patients. Additionally, we did not observe differences in several clinical features related to chronic inflammatory response (BMI/fibrinogen) or metabolic/renal status (glucose, creatinine, DHL). This perception could result from the limited number of observations. However, we observed some differences between pregnant and nonpregnant women, such as a higher serum urea concentration, a longer PT time, and a lower concentration of Ddimer in NP-COVID-19+ than in P-COVID-19+ or P-COVID-19-patients. In pregnancy, such differences occur because of the continuous and physiologic activation of the coagulation system [33]; the longer PT time and lower D-dimer concentration in the NP-COVID-19+ condition could represent the worst adaptation of the pathophysiologic response in SARS-CoV-2 infection. Accordingly, there are few reports of coagulopathy in P-COVID-19+ patients [34]. In contrast, a higher D-dimer concentration has been reported in the general population, which correlates with a high frequency of fatal outcomes [35][36][37]. Interestingly, we observed similar D-dimer concentrations in P-COVID-19+ and P-COVID-19-patients, suggesting that in pregnant women, D-dimer concentration may not be a good predictor for severity or thromboembolic risk [38]. Nevertheless, a larger number of observations within a longitudinal study is needed to assess the usefulness of D-dimer concentration as a predictor in pregnant women with COVID-19.
We observed a lower proportion of HLA-DR+ monocytes in COVID-19+ women with and without pregnancy (Fig 1A), and this lower proportion has also been observed in septic patients with critical conditions or fatal outcomes [39,40]. The lower percentage of HLA-DR + monocytes in COVID-19 could represent the downregulation process of the immune response by SARS-CoV-2, which helps viruses evade immunity, or could be a physiologic mechanism that controls activation of the immune system to avoid overstimulation. More studies are necessary to answer these questions.
An early activation marker (CD69) was also analyzed in our study. We found a similar result for CD69+ cells in both CD4 and CD8 lymphocytes in pregnant and nonpregnant COVID-19 patients (S2 Table). In contrast, a high percentage of CD69+ lymphocytes is observed in pregnant patients with AH1N1 influenza, another respiratory virus challenge that leads to unregulated inflammation in the lungs [41]. These data suggest that depending on the virus that infects pregnant women, different leucocytes become activated, which may require modifications in medical treatment to obtain better results.
Pregnancy is a unique immunological condition, and viral infection represents a major challenge that could alter the immune balance. Innate and acquired responses are participants of a highly regulated immune response in pregnancy [6,8]. CD39 and CD73 are ectoenzymes that sequentially metabolize ATP to adenosine, leading to an anti-inflammatory response [27,32]. Dorneles et al. showed a higher percentage of CD4+CD39+ cells in severe COVID-19 patients than in healthy controls and a lower percentage of CD4+CD73+ cells than in controls [30], suggesting that this could be a useful marker to follow progression in the general population with COVID-19. Our results showed that the percentage of CD39+ or CD73+ B or T cells was not significantly modified by the effects of pregnancy or COVID-19 infection (Fig 1). However, we observed higher percentages of CD39+ monocytes and a lower percentage of CD73+ cells in both pregnant women with and without COVID-19 than in nonpregnant COVID-19+ women (Fig 1). Therefore, pregnant women may modulate inflammation through CD39+ or CD73+ monocytes; these monocytes, as suggested for the general population, could be a potential marker to monitor the evolution of pregnant women hospitalized for COVID-19. Since early growth and remodeling occur in pregnancy, several opportunities to release alarmins (such as ATP) are quite possible; in this regard, CD39-and CD73-positive cells could be useful to maintain the regulation of inflammation. Whether this mechanism could also be involved in limiting the inflammatory response in pregnant women with asymptomatic SARS-CoV-2 infection or in mild COVID-19 has to be elucidated.
Activated leucocytes are a potential source of proinflammatory or regulatory cytokines in the peripheral blood of COVID-19 patients. We determined the percentages of IL-1β+, IL-6 + and IFN-γ leucocytes after 4 hours of culture with or without polyclonal stimulation. CD4 +IL-6+ or IFN-γ+, CD8+IL-6+ or IFN-γ+ lymphocytes and IL-1β+ or IL-6+ monocytes did not reach more than 5% of circulating cells, indicating a low baseline of leucocyte producers for these cytokines in blood. After stimulation with human rIL6, there was no significant increase in IL-1β, IL-6 or IFN-γ lymphocyte or monocyte producers, suggesting that human rIL-6 in COVID-19 patients may not induce the synthesis of proinflammatory cytokines by circulating leucocytes. Polyclonal stimulation with LPS increases the percentage of IL-1β+ or IL-6+ monocytes, and PMA/Ion increases the proportion of CD4+IFN-γ+ and CD8+IFN-γ + T cells, indicating that despite pregnancy or SARS-CoV-2 infection, mononuclear cells are not anergic and retain the ability to express a cytokine response. We observed a trend toward an increase in the percentage of IL-6+ monocytes and a decrease in the percentage of CD4 +IFN-γ+ and CD8+IFN-γ+ lymphocytes in COVID-19 patients, indicating that mononuclear cells support a high concentration of IL-6 and a low response to IFN-γ.
Serological TNF-α, IL-6, CCL4 and IL-4 levels were higher in P-COVID-19+ than in P-COVID-19-, although TNF-α was significantly higher in P-COVID-19+ than in P-COVID-19- (Fig 3A). Interestingly, we also found the highest concentration of IL-4 in the P-COVID-19+ and reached a statistically significant difference with P-COVID-19+ (p = 0.025) and P-COVID-19-(p = 0.012). Our results indicate a similar proinflammatory profile in COVID-19+ patients with or without pregnancy and probably regulated at least partially by IL-4 in pregnant women. Additionally, syncytiotrophoblasts in the human placenta secrete vesicleenclosed microRNAs that could limit viral infections [42]. It has been reported that Let-7 is a miRNA expressed in human cells, let-7a and let-7c inhibit the expression of IL-6, and the increase in the levels of let-7-5p and let-7-3p reduces the expression of IL-1β, IL-8, CCL2 and GM-CSF in cell lines [43]. These results open the possibility that the human placenta regulates cytokine storms in SARS-CoV-2 through miRNAs by the placenta, and more studies are necessary to probe this hypothesis. Some reports have shown that the CXCL10 concentration in serum is associated with poor prognosis in COVID-19+ patients [18]. In contrast, we observed lower CXCL10 concentrations in pregnant COVID-19+ patients than in NP-COVID-19 + patients. This suggests that the immune response in pregnancy is less severe than in the general population.
After polyclonal stimulation, the basal and leucocyte responses showed a similar production of cytokines among the groups (Fig 4), indicating that although COVID-19 peripheral leucocytes have a similar capacity to produce cytokines and suggesting that no anergy or hyperresponse is supported by SARS-CoV-2 infection in leucocytes, hypercytokinemia in COVID-19 could depend not only on leucocytes but also on an alternate source, such as endothelial cells. Human rIL-6 stimulus caused an increase in some cytokines, such as TNF-α and CCL3, indicating that IL-6 signaling favors the synthesis of some cytokines but not the entire set of cytokines related to the so-called cytokine storm. In addition, the response to IL-6 in COVID-19 seems to be similar in the presence or absence of pregnancy, indicating that pregnancy does not necessarily aggravate the proinflammatory responses (at least the leucocyte responses) in COVID-19. To explore whether the leucocyte response is limited by COVID-19, we stimulated cells with LPS or PMA/Ion, and a clear proinflammatory response with cytokines such as TNF-α, CCL3, CCL4, IL-17a, CCL23 and CXCL8 was detected in the supernatant, indicating that leucocytes are not anergic. It has been proposed that immunosuppression rather than a hypercytokine response in COVID-19 could support the pathophysiology [44]. Our results indicate that peripheral blood leucocytes from pregnant women with COVID-19 are capable of expressing a similar response to those from healthy pregnant women.
The main limitation of the present study is the limited number of patients and observations. A greater number of observations is required to reach more certain conclusions. However, the reproducibility and consistency of these results back our analysis. We propose that CD39/CD73 expression in leucocytes, mainly monocytes, could be a candidate to monitor the evolution of COVID-19. On the other hand, unlike in the general population, D-dimer concentrations in pregnant women are not necessarily a marker for severity or thromboembolic risk. Our findings showed that the immune profile in pregnant women was similar to that in nonpregnant women when both were hospitalized for COVID-19. Nevertheless, the slight differences in peripheral leucocyte immunophenotypes suggest that pregnancy elicited pathophysiological mechanisms during COVID-19 infection that require future studies to be clarified.